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DNA polymerase (pol) β catalyzes two reactions at DNA gaps generated during base excision repair, gap-filling DNA synthesis and lyase-dependent 5´-end deoxyribose phosphate removal. The lyase domain of pol β has been proposed to function in DNA gap recognition and to facilitate DNA scanning during substrate search. However, the mechanisms and molecular interactions used by pol β for substrate search and recognition are not clear. To provide insight into this process, a comparison was made of the DNA binding affinities of WT pol β, pol λ, and pol μ, and several variants of pol β, for 1-nt-gap-containing and undamaged DNA. Surprisingly, this analysis revealed that mutation of three lysine residues in the lyase active site of pol β, 35, 68, and 72, to alanine (pol β KΔ3A) increased the binding affinity for nonspecific DNA ∼11-fold compared with that of the WT. WT pol μ, lacking homologous lysines, displayed nonspecific DNA binding behavior similar to that of pol β KΔ3A, in line with previous data demonstrating both enzymes were deficient in processive searching. In fluorescent microscopy experiments using mouse fibroblasts deficient in PARP-1, the ability of pol β KΔ3A to localize to sites of laser-induced DNA damage was strongly decreased compared with that of WT pol β. These data suggest that the three lysines in the lyase active site destabilize pol β when bound to DNA nonspecifically, promoting DNA scanning and providing binding specificity for gapped DNA.

ObjectiveThis research sought to assess the level of COVID-19 preparedness of Emergency Departments (EDs) in Aotearoa New Zealand (NZ) through the views of Emergency Medicine specialists working in District Health Boards around the country. Given the limited experience NZ hospitals have had with SARS-CoV-2, a comparison of current local practice with recent literature from other countries identifying known weaknesses may help prevent future healthcare worker infections in NZ. MethodsA cross-sectional survey by convenience sample of New Zealand Emergency Specialists in November 2020 to evaluate preparedness of engineering, administrative policy, and PPE use. ResultsA total of 137 surveys were completed (32% response rate), revealing heterogeneity in NZ ED clinical work environments in November 2020. More than 10% of emergency specialists surveyed reported no access to negative pressure rooms. N95 fit testing was not done on 15 (11%). Most specialists (86%) work in EDs that cohort patients, about one-third (34%) do not use spotters during PPE doffing, few have policy regarding breaches in PPE, and most do not have required space for physical distancing in non-clinical areas. Initial PPE training, simulations and segregating patients were widespread but appear to be waning with persistent low SARS-CoV-2 prevalence. PPE shortages were not identified in NZ EDs, yet 13% of consultants did not indicate they would use respirators during aerosol generating procedures on COVID-19 patients. Treatment interventions including non-invasive ventilation and high flow nasal cannula were common. Many respondents reported high levels of stress attributable to predicted inadequate staffing and the state of overall preparedness in event of a second wave. ConclusionsNew Zealand emergency specialists identified significant gaps in COVID-19 preparedness, and they have a unique opportunity to translate lessons from other locations into local action. Proactive identification of weaknesses in hospital engineering, policy, and PPE practice in advance of future SARS-CoV-2 endemic transmission would be prudent. What is already known?Aotearoa New Zealand has eliminated COVID-19 community transmission. Recently, trans-Tasman neighbour Australia has controlled SARS-CoV-2 surges which were complicated by significant nosocomial spread and healthcare worker infections. Several recent publications as well as expert recommendations from the Australian Department of Health and Human Services have listed improvements to the Hierarchy of Controls necessary to prevent future outbreaks in hospitals and long-term care facilities. What are the new findings?Survey responses specifically identified breakdowns in engineering, administrative policy and PPE in New Zealand emergency departments (EDs), potentially increasing healthcare worker nosocomial infection risk. As of November 2020, equitable access of all NZ emergency specialists to recommended negative flow rooms, fit testing of N95 masks, and other evidence based policy upgrades to COVID-19 infection prevention and control (IPC) standards are not universal. What do the new findings imply?The experience of local emergency specialists in a rapidly evolving pandemic can identify weaknesses in emergency preparedness previously reported to have increased nosocomial infection risk in similar healthcare environments. The aim of this research was to identify those weaknesses in local NZ emergency department policy, protocols and PPE and further efforts to provide proactive recommendations for system improvement. Finally, the research sought to understand how safe NZ emergency specialists felt during the initial lockdown and provides insight as to the psychological experiences of this vital group of front-line staff.

Ocean sunfish, Mola mola (Linnaeus 1758), is a relatively uncommon display species in public aquariums due to numerous husbandry challenges. A handful of facilities, however, have been able to bring these iconic fish to the broader public. This chapter outlines step-by-step measures for keeping sunfish healthy in aquarium exhibits, highlighting established collection and transportation strategies, various treatment protocols, and the importance of target training to provide specialized food items. By implementing safe handling techniques, improving management strategies and aligning diet design with emerging data on natural dietary shifts, aquarists are learning how to successfully sustain sunfish within enclosures. Partnerships between aquarists and research biologists studying wild sunfish are helping to increase our understanding of sunfish biology while providing aquariums the opportunity to better educate the public on sunfish life history and the functions they serve in the vast oceanic food web.

Packaging of DNA into the nucleosome core particle (NCP) is considered to exert constraints to all DNA-templated processes, including base excision repair where Pol β catalyzes two key enzymatic steps: 5′-dRP lyase gap trimming and template-directed DNA synthesis. Despite its biological significance, knowledge of Pol β activities on NCPs is still limited. Here, we show that removal of the 5′-dRP block by Pol β is unaffected by NCP constraints at all sites tested and is even enhanced near the DNA ends. In contrast, strong inhibition of DNA synthesis is observed. These results indicate 5′-dRP gap trimming proceeds unperturbed within the NCP; whereas, gap filling is strongly limited. In the absence of additional factors, base excision repair in NCPs will stall at the gap-filling step.

DNA polymerase β has two DNA-binding domains that interact with the opposite sides of short DNA gaps. These domains contribute two activities that modify the 5' and 3' margins of gapped DNA during base excision repair. DNA gaps greater than 1 nucleotide (nt) pose an architectural and logistical problem for the two domains to interact with their respective DNA termini. Here, crystallographic and kinetic analyses of 2-nt gap-filling DNA synthesis revealed that the fidelity of DNA synthesis depends on local sequence context. This was due to template dynamics that altered which of the two template nucleotides in the gap served as the coding nucleotide. We observed that, when a purine nucleotide was in the first coding position, DNA synthesis fidelity was similar to that observed with a 1-nt gap. However, when the initial templating nucleotide was a pyrimidine, fidelity was decreased. If the first templating nucleotide was a cytidine, there was a significantly higher probability that the downstream template nucleotide coded for the incoming nucleotide. This dNTP-stabilized misalignment reduced base substitution and frameshift deletion fidelities. A crystal structure of a binary DNA product complex revealed that the cytidine in the first templating site was in an extrahelical position, permitting the downstream template nucleotide to occupy the coding position. These results indicate that DNA polymerase β can induce a strain in the DNA that modulates the position of the coding nucleotide and thereby impacts the identity of the incoming nucleotide. Our findings demonstrate that "correct" DNA synthesis can result in errors when template dynamics induce coding ambiguity.

A first step in processing mitochondrial precursor tRNA (pre-tRNA) is cleavage of the 5’ leader catalyzed by ribonuclease P (RNase P). Human mitochondrial RNase P (mtRNase P) is composed of three protein subunits: mitochondrial RNase P protein (MRPP) 1, 2 and 3. Even though MRPP3 is the metallonuclease subunit responsible for catalysis, cleavage is observed only in the presence of the MRPP1/2 subcomplex. To understand the functional role of MRPP1/2, we reconstituted human mitochondrial RNase P in vitro and performed kinetic and thermodynamic analyses. MRPP1/2 significantly enhances both the catalytic activity and the apparent substrate affinity of mtRNase P. Additionally, pull-down and binding data demonstrate synergy between binding pre-tRNA and formation of a catalytically active MRPP1/2/3 complex. These data suggest that conformational changes in the MRPP1/2-pre-tRNA complex lead to protein-protein or protein-RNA interactions that increase both MRPP3 recognition and cleavage efficiency. This work presents the first kinetic model for human mtRNase P, providing a fundamental framework for the function of MRPP1/2 for recognition and processing of pre-tRNA.

DNA methylation is an epigenetic mark that regulates gene expression in mammals. One method of methylation removal is through ten-eleven translocation-catalyzed oxidation and the base excision repair pathway. The iterative oxidation of 5-methylcytosine catalyzed by ten-eleven translocation enzymes produces three oxidized forms of cytosine: 5-hydroxmethylcytosine, 5-formylcytosine, and 5-carboxycytosine. The effect these modifications have on the efficiency and fidelity of the base excision repair pathway during the repair of opposing base damage, and in particular DNA polymerization, remains to be elucidated. Using kinetic assays, we show that the catalytic efficiency for the incorporation of dGTP catalyzed by human DNA polymerase β is not affected when 5-methylcytosine, 5-hydroxmethylcytosine, and 5-formylcytosine are in the DNA template. In contrast, the catalytic efficiency of dGTP insertion decreases ∼20-fold when 5-carboxycytosine is in the templating position, as compared with unmodified cytosine. However, DNA polymerase fidelity is unaltered when these modifications are in the templating position. Structural analysis reveals that the methyl, hydroxymethyl, and formyl modifications are easily accommodated within the polymerase active site. However, to accommodate the carboxyl modification, the phosphate backbone on the templating nucleotide shifts ∼2.5 A to avoid a potential steric/repulsive clash. This altered conformation is stabilized by lysine 280, which makes a direct interaction with the carboxyl modification and the phosphate backbone of the templating strand. This work provides the molecular basis for the accommodation of epigenetic base modifications in a polymerase active site and suggests that these modifications are not mutagenically copied during base excision repair.

Site-specific DNA binding proteins must search the genome to locate their target sites, and many DNA modifying enzymes have the ability to scan along DNA in search of their substrates. This process is termed processive searching, and it serves to decrease the search time by effectively increasing the DNA binding footprint of a protein. The repertoire of proteins capable of processive searching is expanding, highlighting the need to understand the governing principles behind this fundamental process. Many of the enzymes in the base excision DNA repair pathway are capable of processive searching. Here, we briefly summarize methodology for determining if a protein can scan DNA and highlight the discovery that the base excision repair DNA polymerase β performs a processive search. Elucidation of physical models for DNA searching has also provided a plausible mechanism for pathway coordination during repair. The ability of BER enzymes to transiently sample adjacent DNA sites while bound to their product confers accessibility to downstream enzymes and does not require protein-protein interactions for coordination.

Ribonuclease P (RNase P) catalyzes the cleavage of leader sequences from precursor tRNA (pre-tRNA). Typically, these enzymes are ribonucleic protein complexes that are found in all domains of life. However, a new class of RNase P has been discovered that is composed entirely of protein, termed protein-only RNase P (PRORP). To investigate the molecular determinants of PRORP substrate recognition, we measured the binding affinities and cleavage kinetics of Arabidopsis PRORP1 for varied pre-tRNA substrates. This analysis revealed that PRORP1 does not make significant contacts within the trailer or beyond N−1 of the leader, indicating that this enzyme recognizes primarily the tRNA body. To determine the extent to which sequence variation within the tRNA body modulates substrate selectivity and to provide insight into the evolution and function of PRORP enzymes, we measured the reactivity of the three Arabidopsis PRORP isozymes (PRORP1–3) with four pre-tRNA substrates. A 13-fold range in catalytic efficiencies (104–105 M−1 s−1) was observed, demonstrating moderate selectivity for pre-tRNA substrates. Although PRORPs bind the different pre-tRNA species with affinities varying by as much as 100-fold, the three isozymes have similar affinities for a given pre-tRNA, suggesting similar binding modes. However, PRORP isozymes have varying degrees of cleavage fidelity, which is dependent on the pre-tRNA species and the presence of a 3′-discriminator base. This work defines molecular determinants of PRORP substrate recognition that provides insight into this new class of RNA processing enzymes.

Ribonuclease P (RNase P) is an endonuclease that catalyzes the essential removal of the 5′ end of tRNA precursors. Until recently, all identified RNase P enzymes were a ribonucleoprotein with a conserved catalytic RNA component. However, the discovery of protein-only RNase P (PRORP) shifted this paradigm, affording a unique opportunity to compare mechanistic strategies used by naturally evolved protein and RNA-based enzymes that catalyze the same reaction. Here we investigate the enzymatic mechanism of pre-tRNA hydrolysis catalyzed by the NYN (Nedd4-BP1, YacP nuclease) metallonuclease of Arabidopsis thaliana, PRORP1. Multiple and single turnover kinetic data support a mechanism where a step at or before chemistry is rate-limiting and provide a kinetic framework to interpret the results of metal alteration, mutations, and pH dependence. Catalytic activity has a cooperative dependence on the magnesium concentration (nH = 2) under kcat/Km conditions, suggesting that PRORP1 catalysis is optimal with at least two active site metal ions, consistent with the crystal structure. Metal rescue of Asp-to-Ala mutations identified two aspartates important for enhancing metal ion affinity. The single turnover pH dependence of pre-tRNA cleavage revealed a single ionization (pKa ∼ 8.7) important for catalysis, consistent with deprotonation of a metal-bound water nucleophile. The pH and metal dependence mirrors that observed for the RNA-based RNase P, suggesting similar catalytic mechanisms. Thus, despite different macromolecular composition, the RNA and protein-based RNase P act as dynamic scaffolds for the binding and positioning of magnesium ions to catalyze phosphodiester bond hydrolysis. Background: A protein-only ribonuclease P (PRORP) has been recently discovered. Results: PRORP activity has a single ionization (pKa ∼ 8.7) important for catalysis and a cooperative dependence on Mg2+ (nH = 2). Conclusion: PRORP uses catalytic strategies similar to RNA-dependent RNase P. Significance: These results provide evidence for the mechanistic convergence of two different enzymatic macromolecules (RNA and protein) that perform the same biological function.

DNA repair proteins must locate rare damaged sites within the genome. DNA polymerase β (Pol β), a member of the DNA polymerase X family that is involved in base excision repair, uses a processive hopping search mechanism to locate substrates. This effectively enhances its search footprint on DNA, increasing the probability of locating damaged sites. Processive searching has been reported or proposed for many DNA-binding proteins, raising the question of how widespread or specific to certain enzymes the ability to perform this function is. To provide insight into this question, we compared the ability of three homologous DNA Pol X family members to perform a processive search for 1-nucleotide gaps in DNA using a previously developed biochemical assay. We found that at near-predicted physiological ionic strengths, the intramolecular searching ability of Pol β is at least 4-fold higher than that of Pol μ and ∼2-fold higher than that of Pol λ. Pol β also was able to perform intersegmental transfer with the intersegmental searching ability of Pol β being at least 6- and ∼2-fold higher than that of Pols μ and λ, respectively. Mutational analysis suggested that differences in the N-terminal domains of these polymerases are responsible for the varying degrees of searching competence. Of note, the differences in processive searching ability observed among the DNA Pol X family members correlated with their proposed biological functions in base excision repair and nonhomologous end joining.

Olindiasspp. medusae are found worldwide in sublittoral tropical and sub-tropical coastal regions; their occurrence near shore can result in human envenomation events. While behaviour of medusae and human contact with medusae has been documented for the genus, the hydroid (polyp) phase of theOlindiaslife cycle has eluded investigators for over a century. Given the recent debate among public media and scientific communities that jellyfish blooms are increasing worldwide, there is a growing urgency to understand how and why jellyfish populations bloom. In order to understand jellyfish population dynamics, the asexual benthic phase must be studied to determine when, where, and how juvenile medusae are produced. In this study, husbandry management strategies, including the creation of artificial habitat forOlindias formosusmedusae in aquaria were developed to encourage spawning and larval settlement. The resultant hydroid colony ofOlindias formosuswas discovered in November of 2012, utilizing the natural fluorescence of the medusa as a detection method. A description of the hydroid and early medusa stage is presented. These techniques provide a basis for locatingin situthe benthic hydroid phases within this genus and other fluorescent medusae, the discovery of which may lead to a better understanding of the causative factors for jellyfish blooms.

Ribonuclease P (RNase P) catalyzes the maturation of the 5′ end of precursor-tRNAs (pre-tRNA) and is conserved in all domains of life. However, the composition of RNase P varies from bacteria to archaea and eukarya, making RNase P one of the most diverse enzymes characterized. Most known RNase P enzymes contain a large catalytic RNA subunit that associates with one to 10 proteins. Recently, a protein-only form of RNase P was discovered in mitochondria and chloroplasts of many higher eukaryotes. This proteinaceous RNase P (PRORP) represents a new class of metallonucleases. Here we discuss our recent crystal structure of PRORP1 from Arabidopsis thaliana and speculate on the reasons for the replacement of catalytic RNA by a protein catalyst. We conclude, based on an analysis of the catalytic efficiencies of ribonucleoprotein (RNP) and PRORP enzymes, that the need for greater catalytic efficiency is most likely not the driving force behind the replacement of the RNA with a protein catalyst. The emergence of a protein-based RNase P more likely reflects the increasing complexity of the biological system, including difficulties in importation into organelles and vulnerability of organellar RNAs to cleavage.

DNA polymerase (Pol) β maintains genome fidelity by catalyzing DNA synthesis and removal of a reactive DNA repair intermediate during base excision repair (BER). Situated within the middle of the BER pathway, Pol β must efficiently locate its substrates before damage is exacerbated. The mechanisms of damage search and location by Pol β are largely unknown, but are critical for understanding the fundamental features of the BER pathway. We developed a processive search assay to determine if Pol β has evolved a mechanism for efficient DNA damage location. These assays revealed that Pol β scans DNA using a processive hopping mechanism and has a mean search footprint of ∼24 bp at predicted physiological ionic strength. Lysines within the lyase domain are required for processive searching, revealing a novel function for the lyase domain of Pol β. Application of our processive search assay into nucleosome core particles revealed that Pol β is not processive in the context of a nucleosome, and its single-turnover activity is reduced ∼500-fold, as compared to free DNA. These data suggest that the repair footprint of Pol β mainly resides within accessible regions of the genome and that these regions can be scanned for damage by Pol β.

Jellyfish have become a topic of interest of many marine scientists and managers alike due to their conspicuous socio-economic and environmental impacts. However, our knowledge about their "everyday life" remains limited. While electronic tags (transmitters and loggers) have been extensively used to study marine vertebrates for the past 50 years, tagging is still in its infancy for marine invertebrates and jellyfish in particular. Progress has been hampered by the difficulty and limited knowledge of attaching tags to soft-bodied animals. We argue that there is huge potential to use tagging to gather basic information on the ecology and behaviour of these species. Here, we give an overview of what has been learned so far by deploying tags on jellyfish, and why tagging is an appropriate method to study their behaviour and ecology. We then describe different tagging techniques, their advantages, disadvantages and challenges, and the steps to ensure future successful jellyfish tagging studies.

Ribonuclease P (RNase P) is an essential endonuclease responsible for catalyzing 5’ end maturation in precursor transfer RNAs. Since its discovery in the 1970s, RNase P enzymes have been identified and studied throughout the three domains of life. Interestingly, RNase P is either RNA-based, with a catalytic RNA subunit, or a protein-only (PRORP) enzyme with differential evolutionary distribution. The available structural data, including the active site data, provides insight into catalysis and substrate recognition. The hydrolytic and kinetic mechanisms of the two forms of RNase P enzymes are similar, yet features unique to the RNA-based and PRORP enzymes are consistent with different evolutionary origins. The various RNase P enzymes, in addition to their primary role in tRNA 5’ maturation, catalyze cleavage of a variety of alternative substrates, indicating a diversification of RNase P function in vivo. The review concludes with a discussion of recent advances and interesting research directions in the field.

Ribonuclease P (RNase P) catalyzes the maturation of the 5′ end of tRNA precursors. Typically these enzymes are ribonucleoproteins with a conserved RNA component responsible for catalysis. However, protein-only RNase P (PRORP) enzymes process precursor tRNAs in human mitochondria and in all tRNA-using compartments of Arabidopsis thaliana . PRORP enzymes are nuclear encoded and conserved among many eukaryotes, having evolved recently as yeast mitochondrial genomes encode an RNase P RNA. Here we report the crystal structure of PRORP1 from A. thaliana at 1.75 Å resolution, revealing a prototypical metallonuclease domain tethered to a pentatricopeptide repeat (PPR) domain by a structural zinc-binding domain. The metallonuclease domain is a unique high-resolution structure of a Nedd4-BP1, YacP Nucleases (NYN) domain that is a member of the PIN domain-like fold superfamily, including the FLAP nuclease family. The structural similarity between PRORP1 and the FLAP nuclease family suggests that they evolved from a common ancestor. Biochemical data reveal that conserved aspartate residues in PRORP1 are important for catalytic activity and metal binding and that the PPR domain also enhances activity, likely through an interaction with pre-tRNA. These results provide a foundation for understanding tRNA maturation in organelles. Furthermore, these studies allow for a molecular-level comparison of the catalytic strategies used by the only known naturally evolved protein and RNA-based catalysts that perform the same biological function, pre-tRNA maturation, thereby providing insight into the differences between the prebiotic RNA world and the present protein-dominated world.

This paper reports on a study investigating teachers’ views and beliefs about the relationship between second language (L2) research and practice. Although a gap has been frequently reported between the two, there is little empirical data to show what teachers’ views on this relationship are or how these views and beliefs influence their use of research. A total of 60 teaching English to speakers of other languages (TESOL) teachers in England responded to a questionnaire which sought both qualitative and quantitative data. Results of the data analysis suggest that although their views on research and its usefulness are positive, teachers are mainly sceptical about the practicality and relevance of L2 research. More importantly, they expect research to originate from rather than end in classrooms and maintain that the prime responsibility of bringing research and practice together is to be shared by teacher training programmes and educational policies of the institutions they work in. Our analysis of the d...

What makes employees stay with a particular employer? To find out, we developed a content model of 12 retention factors in the context of previous theory and research and analyzed open-ended responses from 24,829 employees in the leisure and hospitality industry. Our coding of their comments supported the identified framework and revealed that job satisfaction, extrinsic rewards, constituent attachments, organizational commitment, and organizational prestige were the most frequently mentioned reasons for staying. High performers and nonhourly workers were more likely to cite advancement opportunities and organizational prestige as reasons for staying, whereas low performers and hourly employees were more likely to cite extrinsic rewards. These findings highlight the importance of differentiating human resource management practices when the goal is to retain valued employees. © 2009 Wiley Periodicals, Inc.

Despite substantial growth in the service industry and emerging work on turnover consequences, little research examines how unit-level turnover rates affect essential customer-related outcomes. The authors propose an operational disruption framework to explain why voluntary turnover impairs customers' service quality perceptions. On the basis of a sample of 75 work units and data from 5,631 employee surveys, 59,602 customer surveys, and organizational records, results indicate that unit-level voluntary turnover rates are negatively related to service quality perceptions. The authors also examine potential boundary conditions related to the disruption framework. Of 3 moderators studied (group cohesiveness, group size, and newcomer concentration), results show that turnover's negative effects on service quality are more pronounced in larger units and in those with a greater concentration of newcomers.

Protein-only RNase Ps (PRORPs) are a recently discovered class of RNA processing enzymes that catalyze maturation of the 5' end of precursor tRNAs in Eukaryotes. PRORPs are found in the nucleus and/or organelles of most eukaryotic organisms. Arabidopsis thaliana is a representative organism that contains PRORP enzymes (PRORP1, PRORP2 and PRORP3) in both its nucleus and its organelles; PRORP2 and PRORP3 localize to the nucleus and PRORP1 localizes to the chloroplast and the mitochondria. Apart from their identification, almost nothing is known about the structure and function of PRORPs that act in the nucleus. Here, we use a combination of biochemical assays and X-ray crystallography to characterize A. thaliana PRORP2. We solved the crystal structure of PRORP2 (3.2A) revealing an overall V-shaped protein and conserved metallonuclease active-site structure. Our biochemical studies indicate that PRORP2 requires Mg(2+) for catalysis and catalyzes the maturation of nuclear encoded substrates up to 10-fold faster than mitochondrial encoded precursor nad6 t-element under single-turnover conditions. We also demonstrate that PRORP2 preferentially binds precursor tRNAs containing short 5' leaders and 3' trailers; however, leader and trailer lengths do not significantly alter the observed rate constants of PRORP2 in single-turnover cleavage assays. Our data provide a biochemical and structural framework to begin understanding how nuclear localized PRORPs recognize and cleave their substrates.

Ribonuclease P (RNase P) catalyzes removal of 5' end leader sequences from precursor tRNA (pre-tRNA). In most organisms, RNase P is a ribonucleoprotein complex with a catalytic RNA subunit. However...

Demyelination contributes to the loss of function consequent to central nervous system (CNS) injury. Enhanced remyelination through transplantation of myelin-producing cells may offer a pragmatic approach to restoring meaningful neurological function. An unlimited source of cells suitable for such transplantation therapy can be derived from embryonic stem (ES) cells, which are both pluripotent and genetically flexible. In this paper we show that oligodendrocyte cultures can be reliably produced from retinoic acid-induced ES cells and that these oligodendrocytes can myelinate axons in vitro . Methods were further developed for generating highly enriched cultures of oligodendrocytes through an additional culturing step, producing an intermediate “oligosphere” stage. To test whether ES cells can survive, migrate, and differentiate into mature myelin-producing cells in areas of demyelination in the adult CNS, ES cells were transplanted into the dorsal columns of adult rat spinal cord 3 days after chemical demyelination. In the demyelination site, large numbers of ES cells survived and differentiated primarily into mature oligodendrocytes that were capable of myelinating axons. Furthermore, when oligosphere cells were transplanted into the spinal cords of myelin-deficient shiverer ( shi/shi ) mutant mice, the ES cell-derived oligodendrocytes migrated into the host tissue, produced myelin and myelinated host axons. These studies demonstrate the ability of ES cell-derived oligodendrocytes to myelinate axons in culture and to replace lost myelin in the injured adult CNS. Transplantation of ES cells may be a practical approach to treatment of primary and secondary demyelinating diseases in the adult CNS.

This comprehensive case review of hantavirus pulmonary syndrome (HPS) during pregnancy in 5 women characterizes the effect of Sin Nombre virus infection on maternal and fetal outcomes. Histopathologic, serological, and clinical information were evaluated for evidence of vertical transmission. Maternal ages ranged from 20 to 34 years and gestational ages from 13 to 29 weeks. Symptoms, physical findings, and laboratory values other than those related to pregnancy were not noticeably different from those of nonpregnant patients with HPS, although fevers were somewhat lower. One maternal death and 2 fetal losses occurred. Gross, microscopic, and immunohistochemical examination for hantavirus antigen were done on 2 fetal autopsies and 3 placentas showing no evidence of transplacental hantavirus transmission. There was no serological evidence of conversion in the 3 surviving children. Maternal and fetal outcomes of HPS appear similar to those of nonpregnant HPS patients and of pregnant patients with other causes of acute respiratory distress syndrome. No evidence of vertical transmission of Sin Nombre virus was found.

Microorganisms are exposed to a wide variety of exogenous and endogenous chemical agents that alkylate DNA. Escherichia coli cells exhibit an adaptive response that recognizes and repairs alkylated DNA lesions using Ada, AlkA, and AlkB enzymes. Another alkylation response protein, the DNA-binding flavoprotein AidB, was proposed to repair DNA or protect it from chemical alkylating agents, but direct evidence for its role is lacking. Here, AidB was shown to form tight complexes with both flavodoxin and acyl carrier protein. In addition, electron transfer between 1-electron and 2-electron reduced flavodoxin to oxidized AidB was observed, although with very small rate constants. AidB was found to bind to RNA, raising the prospect that the protein may have a role in protection of RNA from chemical alkylation. Finally, the reagent N-methyl-N′-nitro-N-nitrosoguanidine was eliminated as a direct substrate of the enzyme.

Chagas' disease is caused by the protozoan Trypanosoma cruzi which infects 10-15 million people in endemic areas throughout Latin America and is naturally transmitted by insect vectors of the family Reduviidae. Infection can also occur by congenital passage, oral ingestion, laboratory accident, and in organ transplants and blood transfusions. There are 3 life-cycle forms of T. cruzi. Epimastigotes multiply in the midgut of the insect vector, differentiate in the hind-gut into infectious trypomastigotes, and are excreted with the feces or urine after a blood meal. They enter the body by mucous membranes or abraded skin, enter mammalian host cells, escape the phagolysosomal vacuole, and differentiate into amastigotes. Intracellular amastigotes multiply in host cells, redifferentiate to trypomastigotes, and are released upon cell rupture.Acute Chagas' disease can be asymptomatic, or a mild to severe illness. Morbidity can be localized and/or systemic and is usually accompanied by general immunosuppression with blood and tissue parasitemia. It can be fatal. Usually, symptoms and parasitemia decrease within months, followed by a life-long, chronic infection with little morbidity and few apparent parasites. Serologically-positive, chronic asymptomatic patients are termed indeterminate (I). Morbidity develops in 20-30% of chronic patients after 10-30 years and often involves myocardiopathy (Cardiac disease; C), ranging from minor electrocardiographic changes to sudden death by heart failure. Severe “digestive” megasyndromes can also develop. Chagasic myocardial inflammatory infiltrates are associated with cardiac muscle and neuron destruction, followed by progressive fibrotic replacement. Asymptomatic Ipatients who die of unrelated causes have identical (but less intense) lesions.Responses against T. cruzi antigens, their immunoregulation, and responses against related idiotypes (Ids) correlate with the presence of the different clinical forms of the infection. Although there are numerous findings of autoimmune lymphocyte and antibody reactivities in chagasic patients, a causal relationship is always difficult to prove. Chagasic patients' peripheral blood mononuclear cells (PBMC) respond to T. cruzi epimastigote antigens (EPI) with varying vigor. Almost all low responders are Cpatients, and their responses are usually augmented by removal of adherent macrophage suppressor cells or the addition of indomethacin. Chronic I-patients are medium or high responders, and exhibit little adherent cell-mediated immunoregulation. Western blotting studies indicate patients' Ab and cell-mediated responses to EPI show differences between C-and I-patients. PBMC from I-patients responded more often to high molecular weight components (100-150 kD), while both I-and C-patients responded well to moieties between 28-32 and 48-57 kD. All chagasic patients' sera had Abs and PBMC responses to T. cruzi GP57/51 antigen.Chronic Chagas' patients have peripheral blood anti-Id T cells that respond to anti-EPI Abs purified from patients' sera. Some patients' PBMC anti-Id responses to anti-EPI Ids from C-patients (Id-C) are inhibited by chloroquine (Group 1), but some are not inhibited by chloroquine, anti-HLA Class II antigens, or sodium azide (Group 2). Most patients in Group 1 are asymptomatic, but all Group 2 patients have severe disease. Direct (non-processed; non-MHC-presented) stimulation of anti-Id T cells from C-patients by Ids expressed on anti-EPI Abs from C-patients could be immunopathogenic. Anti-Id specific rabbit sera detect Id differences in the anti-EPI Abs from pooled or individual C vs. I-cases. Competitive ELISA assays and Western blot analyses of Abs show that Ids on I-patients' anti-EPI Abs are primary structure expressions, while Ids on C-patients' anti-EPI Abs are defined by intact Ab molecules.

Murine embryonic stem cells were induced to differentiate into neural lineage cells by exposure to retinoic acid. Approximately one million cells were transplanted into the lesion site in the spinal cords of adult rats which had received moderate contusion injuries 9 days previously. One group received transplants of cells genetically modified to over-express bcl-2, which codes for an anti-apoptotic protein. A second group received transplants of the wild-type ES cells from which the bcl-2 line was developed. In the untransplanted control group, only medium was injected. Locomotor abilities were assessed using the Basso, Beattie and Bresnahan (BBB) rating scale for 6 weeks. There was no incremental locomotor improvement in either transplant group when compared to control over the survival period. Morbidity and mortality were significantly more prevalent in the transplant groups than in controls. At the conclusion of the 6-week survival period, the spinal cords were examined. Two of six cords from the bcl-2 group and one of 12 cords from the wild-type group showed gross evidence of abnormal growths at the site of transplantation. No similar growth was seen in the control. Pathological examination of the abnormal cords showed very large numbers of undifferentiated cells proliferating at the injection site and extending up to 1.5 cm rostrally and caudally. These results suggest that transplanting KD3 ES cells, or apoptosis-resistant cells derived from the KD3 line, into the injured spinal cord does not improve locomotor recovery and can lead to tumor-like growth of cells, accompanied by increased debilitation, morbidity and mortality.

Traditionally, treatment of spinal cord injury seemed frustrating and hopeless because of the remarkable morbidity and mortality, and restricted therapeutic options. Recent advances in neural injury and repair, and the progress towards development of neuroprotective and regenerative interventions are basis for increased optimism. Neural stem cells have opened a new arena of discovery for the field of regenerative science and medicine. Embryonic stem (ES) cells can give rise to all neural progenitors and they represent an important scientific tool for approaching neural repair. The growing number of dedicated regeneration centers worldwide exemplifies the changing perception towards the do-ability of spinal cord repair and this review was born from a presentation at one such leading center, the Kentucky Spinal Cord Injury Research Center. Current concepts of the pathophysiology, repair, and restoration of function in the damaged spinal cord are presented with an overlay of how neural stem cells, particularly ES cells, fit into the picture as important scientific tools and therapeutic targets. We focus on the use of genetically tagged and selectable ES cell lines for neural induction and transplantation. Unique features of ES cells, including indefinite replication, pluripotency, and genetic flexibility, provide strong tools to address questions of neural repair. Selective marker expression in transplanted ES cell derived neural cells is providing new insights into transplantation and repair not possible previously. These features of ES cells will produce a predictable and explosive growth in scientific tools that will translate into discoveries and rapid progress in neural repair.

Demyelination contributes to the loss of function consequent to central nervous system (CNS) injury. Optimizing remyelination through transplantation of myelin-producing cells may offer a pragmatic approach to restoring meaningful neurological function. An unlimited source of cell suitable for such transplantation therapy can be derived from embryonic stem (ES) cells, which are both pluripotent and genetically flexible. Here we review work from our group showing that neural precursor cells can be derived from ES cells and that transplantation of these cells into the injured spinal cord leads to some recovery of function. We have further examined and optimized methods for enriching oligodendrocyte differentiation from ES cells. ES cell-derived oligodendrocytes are capable of rapid differentiation and myelination in mixed neuron/glia cultures. When transplanted into the injured spinal cord of adult rodents, the neural-induced precursor cells are capable of differentiating into oligodendrocytes and myelinating host axons. The role of myelination and remyelination will be discussed in the context of regeneration strategies.

Demyelination contributes to the loss of function consequent to central nervous system (CNS) injury. Optimizing remyelination through transplantation of myelin-producing cells may offer a pragmatic approach to restoring meaningful neurological function. An unlimited source of cell suitable for such transplantation therapy can be derived from embryonic stem (ES) cells, which are both pluripotent and genetically flexible. Here we review work from our group showing that neural precursor cells can be derived from ES cells and that transplantation of these cells into the injured spinal cord leads to some recovery of function. We have further examined and optimized methods for enriching oligodendrocyte differentiation from ES cells. ES cell-derived oligodendrocytes are capable of rapid differentiation and myelination in mixed neuron/glia cultures. When transplanted into the injured spinal cord of adult rodents, the neural-induced precursor cells are capable of differentiating into oligodendrocytes and myelinating host axons. The role of myelination and remyelination will be discussed in the context of regeneration strategies.

This article discusses the relationship between disasters and infections. Infections that are reviewed include those resulting from (1) a breakdown of the usual mechanisms of infection control, (2) the introduction or emergence of pathogens, and (3) the movement of populations into new areas. Components of infectious-disease surveillance and disaster teams are detailed.

Study objective: To define the contribution of domestic violence (DV) to homicides in women in New Mexico and to examine differences in ethnicity, mechanism, previous documented injuries, incidence of sexual assault, and use of alcohol or illicit drugs between DV- and non-DV-related homicides. Methods: We carried out a retrospective analysis of reports of the state office of the medical investigator (OMI) reports for all female homicides from 1990 to 1993 in New Mexico. A homicide was defined as being related to DV if the perpetrator was a current or former male intimate partner. The χ 2 and Mann-Whitney tests were used to analyze data. Results: The OMI investigated 134 homicides in women for an overall fatality rate of 4.3 per 100,000. A male intimate partner was the perpetrator in 62 cases (46%). The rate of DV homicide among American Indians (4.9 per 100,000) was significantly higher than that among Hispanics (1.7) and non-Hispanic whites (1.8) (RR=2.8; 95% confidence interval [CI], 1.5 to 5.1). Firearms were almost two times as likely to be used in DV homicides as in non-DV homicides (RR=1.8; 95% CI, 1.2 to 2.6). Evidence of old injuries was found more often in DV homicide cases (35.5%) than in non-DV cases (8.3%) (RR=4.3; 95% CI, 1.8 to 9.8). The presence of alcohol or other drugs was higher among non-DV homicide victims (69%) than DV homicide victims (54.3%) (P=.03). Conclusion: American Indian women are at particularly high risk of homicide, including DV homicide. Firearms were overrepresented in DV homicides, suggesting that removing firearms from the homes of previous DV perpetrators would be a useful public health strategy. Alcohol or illicit drugs were found in approximately two thirds of New Mexico women who were victims of homicide. The high prevalence of history of previous injuries among DV homicide victims indicates that early identification of DV victims in the emergency department and other health care settings is an important point of intervention. [Arbuckle J, Olson L, Howard M, Brillman J, Anctil C, Sklar D: Safe at home? Domestic violence and other homicides among women in New Mexico. Ann Emerg Med February 1996;27:210-215.]

Glucocorticoid excess causes insulin resistance and hypertension. Hepatic expression of PPARalpha (Ppara) is required for glucocorticoid-induced insulin resistance. Here we demonstrate that afferent fibers of the vagus nerve interface with hepatic Ppara expression to disrupt blood pressure and glucose homeostasis in response to glucocorticoids. Selective hepatic vagotomy decreased hyperglycemia, hyperinsulinemia, hepatic insulin resistance, Ppara expression, and phosphoenolpyruvate carboxykinase (PEPCK) enzyme activity in dexamethasone-treated Ppara(+/+) mice. Selective vagotomy also decreased blood pressure, adrenergic tone, renin activity, and urinary sodium retention in these mice. Hepatic reconstitution of Ppara in nondiabetic, normotensive dexamethasone-treated PPARalpha null mice increased glucose, insulin, hepatic PEPCK enzyme activity, blood pressure, and renin activity in sham-operated animals but not hepatic-vagotomized animals. Disruption of vagal afferent fibers by chemical or surgical means prevented glucocorticoid-induced metabolic derangements. We conclude that a dynamic interaction between hepatic Ppara expression and a vagal afferent pathway is essential for glucocorticoid induction of diabetes and hypertension.